The present invention relates to a method for readily and reliably detecting single base substitutions in nucleic acid polymers, particularly those substitutions located in the highest melting domain of duplexed nucleic acids.
Melting is defined as the dissociation of the hydrogen bonds that hold double-stranded nucleic acid polymers together. Nucleic acid melting proceeds under equilibrium conditions as a series of relatively abrupt transitions of portions of the polymer (i.e., domains) from helix to random chain. The number of base pairs cooperating to define each domain is determined by the nucleotide sequence. By analysis of the sequence of a DNA molecule, it could be predicted under what conditions the different domains would melt. The lowest melting domain (LMD) comprises those portions of the polymer which melt under the mildest conditions, whereas the highest melting domain (HMD) comprises those that require the most severe conditions to melt.
Denaturing gradient gel electrophoresis for the detection of mutations in DNA molecules was originally developed by Lerman et al., Ann. Rev. Biophys. Bioeng. 13:399-423, 1984. This technique was based on the observation that the electrophoretic mobility of DNA in polyacrylamide gels is sensitive to the secondary structure of the molecule; i.e. helicity, partial melting or complete melting with strand dissociation. Partially melted nucleic acid polymers consisting of double helical regions and disordered single-stranded regions move much more slowly than complete double helixes or completely melted molecules.
The Lerman et al. technique, which makes it possible to resolve complex mixtures of nucleic acid polymers, utilizes a gradient of denaturing solvent in a uniform polyacrylamide gel, run and maintained at the temperature of incipient DNA melting. The solvent gradient provides the equivalent of a shallow linear temperature increase so that dissociation occurs in successively higher melting domains of the DNA.
Recently, further modifications of the Lerman et al. technique have allowed the detection of mutations in genomic RNA of influenza A viruses by analysis of RNA-RNA and RNA-DNA heteroduplexes. See e.g. Smith et al., Virology 150:55-64, 1986. Heteroduplexes were made by hybridizing virion RNA with SP6- or M13-derived cDNA probes of varying length, followed by Sl nuclease digestion to remove unhybridized probe. Duplexes moved through the denaturing acrylamide gel at mobilities determined by molecular weight until they migrated into a denaturant concentration sufficient to melt the duplex. As melting of each domain occurred, the electrophoretic mobility of the fragment abruptly changed. For example, partial strand separation due to melting of low melting domains of a duplex resulted in an abrupt mobility decrease which produced sharp focusing of the bands on the gels. Moreover, differences were observed in melting behavior between perfectly base-pair matched and mismatched low melting domains, that resulted in separation on the gel.
Melting of the highest melting domain (HMD) in a fragment led to strand dissociation, and under these conditions the resolving power of the gel was lost. Accordingly, it was not possible to distinguish perfectly matched from mismatched duplexes if the mismatch occurred within the HMD.
It was recently shown that detection of base substitutions in the higher melting domains of double-stranded DNA (dsDNA) molecules was greatly improved by the addition of a GC-rich nucleotide sequence at one end of the molecule which functioned as a clamp. See, e.g. Myers et al., Nucleic Acids Res. 13:3131-3145, 1985. However, the incorporation of a GC clamp requires cloning the sequence to be analyzed. Also, it is not easily applicable to the analysis of mutations in RNA molecules.
It has now been found that base substitutions in the highest melting domain of double-stranded nucleic acid polymers, including DNA-DNA, DNA-RNA and RNA-RNA, can be readily detected using gel electrophoresis by first melting the duplexes in solution and then using the gel system to monitor for strand dissociation.